Island plants with newly discovered reproductive traits have higher capacity for uniparental reproduction, supporting Baker’s law

Uniparental reproduction is advantageous when lack of mates limits outcrossing opportunities in plants. Baker’s law predicts an enrichment of uniparental reproduction in habitats colonized via long-distance dispersal, such as volcanic islands. To test it, we analyzed reproductive traits at multiple hierarchical levels and compared seed-set after selfing and crossing experiments in both island and mainland populations of Limonium lobatum, a widespread species that Baker assumed to be self-incompatible because it had been described as pollen-stigma dimorphic, i.e., characterized by floral morphs differing in pollen-surface morphology and stigma-papillae shape that are typically self-incompatible. We discovered new types and combinations of pollen and stigma traits hitherto unknown in the literature on pollen-stigma dimorphism and a lack of correspondence between such combinations and pollen compatibility. Contrary to previous reports, we conclude that Limonium lobatum comprises both self-compatible and self-incompatible plants characterized by both known and previously undescribed combinations of reproductive traits. Most importantly, plants with novel combinations are overrepresented on islands, selfed seed-set is higher in islands than the mainland, and insular plants with novel pollen-stigma trait-combinations disproportionally contribute to uniparental reproduction on islands. Our results thus support Baker’s law, connecting research on reproductive and island biology.


Table S2 Results of GLMMs.
Table S3 Raw data of pollen and stigma traits (TYPICAL: pollen-stigma combinations previously described in Limonieae; PREV.UNDESCRIBED: pollen-stigma combinations that are new in Limonieae) (see separate file).

Methods & Results S1: Effect of developmental stage (i.e., age) and position on variation of stigma traits within plants
We observed intra-plant diversity of stigma traits (Figs 3-5).It is well known that developmental stage and position within inflorescences can affect the expression of floral traits 1,2 .In Limonium, corolla, stigmas, and anthers wither within less than 24 hours after flower opening.For morphological analyses, we collected freshly opened flowers at the same day, if possible (see Table S3a).Therefore, all experimental flowers were at the same developmental stage when analyzed.Within spikes and spikelets, flowers bloom from the proximal to the distal part.Consequently, the later blooming flowers occur more distal within a spike or spikelet.If positional effects affected intra-plant variability of stigma traits, we would expect stigma traits to differ systematically in early vs. late blooming flowers.To test this prediction, we randomly sowed a second batch of seeds from each of the five island populations in July 2012, repotted six seedlings per population, and raised a total of 30 plants as described in the main section of the paper.One plant died.We thus harvested a total of 264 flowers from 29 individuals over a period of 13 to 84 days (details in Table S3b).Ten flowers had less than three intact stigmas and were therefore excluded from the analysis.From the remaining 254 flowers, we determined stigma traits as described in the main section of the paper.Five out of 29 plants had homogeneous stigma traits (cob = 3 plants; pap = 2 plants), while 24 plants had variable stigma traits.In the latter group of plants, stigma traits did not produce consistent patterns of trait variation between early and late blooming flowers (Fig. S6), suggesting that positional effects should not have appreciably altered the results.

Methods & Results S2: Effects of timing of manual pollination and pollen type (one vs. two) on outcrossed seed-set
Effect of timing -Plants from populations I-1, I-4, I-5, M-1, M-2, and M-4 produced considerably more flowers than plants from populations I-2, I-3, M-3, and M-5.Therefore, the cross treatment (CROSS) could only be carried out on plants from the former set of populations (Fig. S3b).In addition, plants were cross-pollinated up to 19 days after they were manually selfed (islands: median = 16.5 days; mainland: median = 11.0 days; Table S4), which could affect the success of selfed vs. outcrossed seed formation 3,4 .If this time lag negatively affected outcrossed seed-set, we would expect a negative and significant correlation between seed-set of cross-pollinated flowers and the time lag between the two manual pollination treatments (number of days between SELFGEITO and CROSS) in BOTH localities.To test this prediction, we calculated Spearmans rho between outcrossed seed-set and time lag for mainland and island plants.The correlation between seed set and time lag was not significant neither for island (Spearmans rho = -0.035,N = 28, P = 0.860) nor mainland plants (Spearmans rho = 0.017, N= 29, P = 0.930) indicating that the time lag between SELFGEITO and CROSS should not have appreciably altered the results.
Effect of pollen type -If pollen and stigma trait combinations predicted SI, stigmas should be more compatible with one of the two dimorphic pollen types.In the manual pollination experiment, ten out of 57 cross-pollinated plants occurred in the pollen-dimorphic population I-1, while 47 plants occurred in pollen-monomorphic populations (I-4: 10 plants; I-5: 8 plants; M-1: 9 plants; M-2: 10 plants; and M-4: 10 plants; Figs 5d, S3b).Since we did not record the pollen type of the pollen donors in the CROSS treatment, we might have pollinated flowers with either A pollen, or B pollen, or both in the pollen-dimorphic population I-1.If the two pollen types triggered incompatibility reactions of different strengths, we would expect outcrossed seed-set to vary more in the pollen-dimorphic I-1 than the five pollen-monomorphic populations (I-4, I-3, M-1, M-2, and M-4).To test this prediction, we estimated for each population the variance in outcrossed seeds-set and tested with a one-tailed one-sample Wilcoxon Signed Rank Test, whether outcrossed seed-set varied significantly more in pollen-dimorphic than pollen-monomorphic populations.The variance of seed-set after outcrossing treatment in the pollen-dimorphic population I-1was σ 2 = 0.005, while it was 0.033 ± 0.014 (M ± SE) in the pollen-monomorphic populations.Hence, the variance of seed-set after outcrossing treatment in the pollen-dimorphic population I-1 was significantly smaller than the variance of seed-set after outcrossing treatment across all pollen-monomorphic populations (N = 5, Z = 2.032, P = 0.021).However, since this result is in the opposite direction of our expectation (i.e., we expected the variance of seed-set after outcrossing treatment in I-1 to be larger than the variance of seed-set after outcrossing treatment across all pollen-monomorphic populations) the result is non-significant (P = 0.979).This finding indicates that the presence of two vs. one pollen type in the populations should not have appreciably altered our results.Table S3 Raw data of pollen and stigma traits (TYPICAL: pollen-stigma combinations previously described in Limonioideae; PREV.UNDESCRIBED: pollen-stigma combinations that are new in Limonioideae) (see separate file).

Figure S2
Figure S2Flower and Inflorescence structure in Limonium lobatum.

Figure S3
Figure S3Sampling and number of plants and flowers used to study pollen and stigma traits and seed set.

Figure S6
Figure S6Temporal variation of stigma traits.

3 .
Diversity of stigmatic papillae types discovered in Limonium lobatum.Stigmas with various combinations (var stigmas) of cob (flat shape), pap (protruding shape), and int (intermediate shape) papillae in the same stigma and among stigma of the same gynoecium.Arrows illustrate shapes of cob, pap and int papillae.

Figure S2 .
Figure S2.Flower (a) and inflorescence (b) structures in Limonium lobatum.(a) Left: dissected out gynoecium consisting of the ovary with one ovule, the five styles, and the five filiform (linear) stigmas; Right: dissected out corolla and androecium depicting two out of the five filaments and anthers.(b) From left to right: a spike comprises 6-12 spikelets; a spikelet comprises two to three flowers; a flower comprises a single ovule and produces one seed.Scale bar = 1 cm (see also species description by Brullo & Erben 5 ).

Figure S3 .
Figure S3.Sampling and number of plants and flowers used to characterize (a) pollen and stigma traits and (b) seed set of Limonium lobatum in five populations from the Canary Islands (I-1 to I-5, red quadrilaterals), five populations from the Iberian mainland (M-1 to M-5; green quadrilaterals), and one population from the Dead Sea (DS; blue quadrilaterals).Abbreviations: PL, plant; FL, flower; −, no data available; and abbreviations of populations as reported in legend of Fig. 2.

Figure S4 .
Figure S4.Pollen-stigma interactions within stigmas of Limonium lobatum.Previous studies demonstrated that an incompatibility system prevents the germination of pollen grains or, if pollen grains germinate, the penetration of the stigmatic surfaces by the pollen tubes in plants with cob stigmas and A pollen (A/cob) and plants with pap stigmas and B pollen (B/pap; Fig. 1) 6,7 .(a) Pictures of A pollen on cob stigmas (left panel) and B pollen on pap stigmas (right panel).(b) Stages of pollen-stigma interactions: mean values and standard errors of relative number of self-pollinated flowers per plant with germinated pollen grains (GERMINATION) and pollen tubes that have penetrated the stigmatic surfaces (PENETRATION).For definitions of pollen and stigma traits, see Fig.4.

4
Figure S4.Pollen-stigma interactions within stigmas of Limonium lobatum.Previous studies demonstrated that an incompatibility system prevents the germination of pollen grains or, if pollen grains germinate, the penetration of the stigmatic surfaces by the pollen tubes in plants with cob stigmas and A pollen (A/cob) and plants with pap stigmas and B pollen (B/pap; Fig. 1) 6,7 .(a) Pictures of A pollen on cob stigmas (left panel) and B pollen on pap stigmas (right panel).(b) Stages of pollen-stigma interactions: mean values and standard errors of relative number of self-pollinated flowers per plant with germinated pollen grains (GERMINATION) and pollen tubes that have penetrated the stigmatic surfaces (PENETRATION).For definitions of pollen and stigma traits, see Fig.4.
Figure S4.Pollen-stigma interactions within stigmas of Limonium lobatum.Previous studies demonstrated that an incompatibility system prevents the germination of pollen grains or, if pollen grains germinate, the penetration of the stigmatic surfaces by the pollen tubes in plants with cob stigmas and A pollen (A/cob) and plants with pap stigmas and B pollen (B/pap; Fig. 1) 6,7 .(a) Pictures of A pollen on cob stigmas (left panel) and B pollen on pap stigmas (right panel).(b) Stages of pollen-stigma interactions: mean values and standard errors of relative number of self-pollinated flowers per plant with germinated pollen grains (GERMINATION) and pollen tubes that have penetrated the stigmatic surfaces (PENETRATION).For definitions of pollen and stigma traits, see Fig.4.

Figure
Figure S5 (previous page).Plots depicting Pearson Residual vs. Predicted Probability of Seed production, as estimated from generalized linear mixed effects models (GLMM), testing whether seed-set differed (a) among pollination treatments (autonomous selfing, geitonogamous selfing, and cross-pollination) and plant types (PREV.UNDESCRIBED vs. TYPICAL, subdivided into pollen-stigma combinations previously described as selfincompatible (A/cob & B/pap) and self-compatible (B/cob & A/pap), (b) among pollination treatments (autonomous selfing, geitonogamous selfing, and cross-pollination) and location (island and mainland), and (c) between manually selfed plant types (previously undescribed & previously described) on the islands.

Figure S6 .
Figure S6.Temporal variation of stigma traits of individual island plants of Limonium lobatum.Abbreviations: pap = all stigmas of a flower with uniform protruding stigmatic papillae cells; cob = all stigmas of a flower with uniformly flat stigmatic papillae cells, var = either all stigmas of a flower with variable shapes of papillae cells or at least one stigma different from the others (see also Fig.4).Abbreviations of populations as reported in legend of Fig.2.